Issued for: Compact Dry EC Method for the Enumeration of ......page 2 (12) PRINCIPLE OF THE METHOD ....

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NMKL - NordVal International c/o Institute of Marine Research P.O. box 1870 Nordnes, N-5817 Bergen, Norway www.nmkl.org

Manufactured by: Supplied by: Nissui Pharmaceutical Co.Ltd, 3-23-9 Ueno, Taito-ku, Tokyo, 110-8736 Japan

HyServe GmbH & Co. KG, Hechenrainerstr 24, 82449 Uffing, Germany

Compact Dry EC The performance of this has been compared to the reference methods:

• ISO 16649-2:2001: “Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli. Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide.” and

• ISO 4832:2006: “Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coliforms. Colony-count technique.”

The validation studies have been conducted by Campden, UK, according to t ISO 16140-2:2016, and concludes Compact Dry EC provide equivalent results to the reference methods. The production of Compact Dry Ec is certified according to ISO 9001 and ISO 13485.

Date: 1/12 2020

Yours sincerely,

Hilde Skår Norli Eystein Oveland Chair of NordVal International NMKL Secretary General

Issued for: Compact Dry EC Method for the Enumeration of Escherichia coli and coliforms

NordVal No: 036 First approval date: 1 December 2008 Renewal date: 1 December 2020 Valid until: 1 December 2022

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PRINCIPLE OF THE METHOD Compact Dry EC is a ready-to-use dry chromogenic plate for enumeration of E.coli and coliforms. Pre-treat the samples according to ISO 6687 or NMKL 91. The medium contains two kinds of chromogenic enzyme substrates: Magenta-Gal and X-Gluc. E.coli forms blue colonies. The total coliform group count is the sum of both the red and blue colonies. An aliquot of 1 ml of an appropriate dilution is plated onto Compact Dry EC plate. The incubation conditions tested in the study were 37 ± 1°C for 24 ± 2h. FIELD OF APPLICATION The method has been tested on enumeration of Escherichia coli and coliforms in a broad range of foods. HISTORY In 2007, the method was validated according to the ISO 16140:2003. Every two years until 2018 the method has been renewed without any additional studies. In 2018 a renewal study was performed to comply with the requirements for relative trueness and accuracy profile in the new standard ISO 16140-2:2016. As the design of the Inter-laboratory study (ILS) is the same for the 2003 and 2016 versions of ISO16140, the data from the ILS data of 2007 are re-evaluated using the new statistical approach outlined in ISO16140-2:2016. COMPARISON STUDIES Relative trueness study The trueness study is a comparative study between results obtained by the reference method and the results of the alternative method. Different categories, types and items were tested as shown in Table 1 below. Table 1 – Categories and types tested Category Types No. of

samples

Milk and dairy products

Dry milk product e.g. milk powder, powder for milk based desserts, dried infant formula

5

Dairy products e.g. ice-cream, yogurts, cream, hard cheese, soft cheese, raw milk cheese

5

Pasteurised milk products e.g. skimmed, semi-skimmed, full fat and flavoured milks

5

Fresh produce and fruits

Cut ready to eat fruit e.g. fruit mixes, fruit juices 5 Cut ready to eat vegetables e.g. Bagged pre-cut salads and shredded carrot, cabbage, vegetable juices

5

Leafy greens/Sprouts e.g. soy, mung, alfalfa, 5 Raw poultry and meats (Combined category raw/ RTC meats and poultry)

Fresh poultry cuts e.g. turkey breast, turkey fillet 5 Fresh mince e.g. lamb, beef, pork 5 Processed ready to cook e.g. frozen patties, marinated kebabs, seasoned chicken breasts

5

Ready to eat foods (Combined category RTE/RTRH meats and poultry)

Ready to eat poultry e.g. turkey fillet, chicken sausage, pate 5 Cooked fish products e.g. prawns, terrine, pate, smoked fish 5 Cooked meat e.g. ham, salami, pate, corned beef 5

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87654321

1.5

1.0

0.5

0.0

-0.5

-1.0

Mean of log10 cfu/g

Diffe

renc

e (a

ltern

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e - r

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)

95% limit

95% limit

Identity

Fresh produce and fruitsMilk and dairymulti component foodsRaw meat and poultryready to eat foods

All Categories– – Bias

Multi component foods or meal components

Ready to re-heat refrigerated food e.g. cooked chilled foods, rice and pasta, products

5

Ready to re-heat food frozen e.g. fries, pizza 5 Composite foods with substantial raw ingredients e.g. pasta salads, sandwiches, deli-salads

5

The relative trueness is illustrated by the use of a Bland-Altman plot, i.e. the difference (bias) between paired samples analysed with the reference method and the alternative method respectively, plotted against the mean values obtained by the reference method. In the plot, Upper and Lower limits are included as the bias ± 2 times the standard deviation of the bias. The Bland-Altman Plot in Figure 1, illustrates the difference obtained in the enumeration of E.coli and total count by the alternative and the reference method, respectively. Figure 1 Bland-Altman Plot of the enumeration of E.coli and total count in foods E. coli:

7654321

1.0

0.5

0.0

-0.5

-1.0

Mean of log10 cfu/g

Diffe

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e - r

efer

ence

)

95% limit

95% limit

Identity

Fresh produce and fruitsMilk and dairymulti component foodsRaw meat and poultryready to eat foods

All Categories– – Bias

Coliforms:

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It is expected that no more than 1 in 20 data values will lie outside the 95% confidence levels (upper limit and lower limits). For E.coli: For ‘All Categories’ there are five in 75 values which lie outside the CLs. This is a little more than the expectation of less than one in 20. There was no identifiable trend in the data outside the CLs, which covered 4 different food categories, 4 different inoculated strains and 3 different seeding/spiking protocols. For coliforms: For ‘All Categories’ there are six in 75 values which lie outside the CLs. This is a little more than the expectation of less than one in 20. Of the six points outside of the CLs, the data covered 3 different food categories, and 3 different inoculated strains.

ACCURACY PROFILE The accuracy profile study is a comparative study between the results obtained by the reference method and the results of the alternative method. Each item used were artificially contaminated obtaining three target levels; low (102 cfu/g), medium (104 cfu/g) and high (106 cfu/g). Five test portions of each level of each item were analysed, resulting in 150 samples. The tested categories, types, items and inoculated strains are provided in the Table 2. Table 2 - Categories, types and food items

Category Types Strains – E.coli Strains - Coliforms

Items

Dairy products Pasteurised dairy products

E. coli CRA 1476 from dried milk

E. coli CRA 1476 from dried milk

Pasteurised cream

E.coli NCTC 8008 Enterobacter agglomerans CRA 5613 from milk powder

Cream cheese

Fruits and vegetables Fresh produce

E.coli ATCC 25922 E.hermanii CRA 7477 from sesame seeds

Ready to cook Vegetable preparation

E. coli NCIMB 700555

Citrobacter amalonaticus CRA 7458 from beansprouts

Vegetable juice

Raw poultry and meats (Combined category raw/ RTC meats and poultry)

Fresh meat

E. coli CRA 16041 from raw ground mince

Enterobacter aerogenes NCTC 10006 Pork mince

E. coli CRA 1593 from poultry

Citrobacter freundii NCTC 9750 Raw bacon

Ready to eat foods (Combined category RTE/RTRH meats and poultry)

Cooked fish products e.g. prawns

E.coli CRA 2003 isolated from fish

E.coli CRA 2003 from fish

E.coli CRA 1968 isolated from lamb

Klebsiella oxytoca ATCC 15926 Fish pate

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Multi component foods

Composite foods with raw ingredients

E.coli CRA 16044 isolated from beef

Enterobacter agglomerans CRA 5513 from skimmed milk powder

Sandwiches

E.coli CRA 1265 dried foods

E. adecarboxylata CRA 5501 from skimmed milk powder

Cooked chilled rice

The total number of samples analysed for both E.coli and Coliforms with both methods were 150. The statistical results and the accuracy profiles are provided in the Figures 2 to 6. Figure 2 Dairy products E.Coli: Coliforms:

Sample Name Reference central value Bias Lower β-ETI Upper β-ETI

β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

107 45 75 342 322 1,78 0,222 -0,140 0,584 NO YES

299 41 22 277 230 2,34 -0,112 -0,474 0,250 YES YES

314 346 25 63 328 3,66 0,182 -0,180 0,545 NO YES

198 154 231 338 254 4,20 -0,204 -0,566 0,158 NO YES

58 141 172 83 109 5,45 0,316 -0,046 0,679 NO YES

130 233 94 162 100 6,32 -0,067 -0,429 0,295 YES YES

Reference method

Alternative method

SD Repeatability 0,184 0,251 +/- 0,736

SD repeatability of reference method <= 0.125 Final AL

(Food) Category DAIRY(Food) Type PASTEURISED DAIRY

NO

-1,00

-0,80

-0,60

-0,40

-0,20

0,00

0,20

0,40

0,60

0,80

1,00

0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00

Bias

Reference Median

PASTEURISED DAIRY

Bias

β-ETI

AL = +/- 4SDr

Sample Name Reference central value Bias Lower β-ETI Upper β-ETI

β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

142 21 248 188 53 1,85 -0,067 -0,256 0,122 YES YES

107 45 75 342 322 3,11 -0,114 -0,303 0,075 YES YES

31 10 97 13 273 3,88 -0,049 -0,238 0,140 YES YES

314 346 25 63 328 3,97 0,282 0,093 0,471 YES YES

196 229 57 102 259 5,66 0,000 -0,189 0,189 YES YES

58 141 172 83 109 5,76 0,198 0,009 0,387 YES YES

Reference method

Alternative method

SD Repeatability 0,132 0,131 +/- 0,500


(Food) Category dairy(Food) Type pasteurised dairy

NO

-0,60

-0,40

-0,20

0,00

0,20

0,40

0,60

0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00

Bias

Reference Median

pasteurised dairy

Bias

β-ETI

AL = +/- 0.5

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Figure 3 Fruit and vegetable products E.Coli:

Sample Name Reference Central value Bias Lower β-ETI Upper β-ETI

β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

164 79 181 213 305 2.00 0.176 -0.027 0.380 YES YES

119 121 176 150 91 2.75 0.155 -0.049 0.358 YES YES

276 129 67 3 174 4.26 0.087 -0.116 0.291 YES YES

116 104 88 201 105 4.40 0.215 0.011 0.418 YES YES

173 98 44 127 52 6.08 0.243 0.039 0.447 YES YES

140 326 66 190 343 6.38 0.138 -0.065 0.342 YES YES

Reference method

Alternative method

SD Repeatability 0.132 0.141 +/- 0.500

SD repeatability of reference method <= 0.125

NO

Final AL

(Food) Category(Food) Type

fruit and vegfresh produce

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Bias

Reference Median

fresh produce

Bias

β-ETI

AL = +/- 0.5

Coliforms:


β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

8 17 327 246 37 1.65 -0.255 -0.491 -0.020 YES YES

264 252 139 300 294 2.15 -0.146 -0.381 0.089 YES YES

312 301 64 267 340 3.46 -0.248 -0.483 -0.012 YES YES

236 92 223 9 158 3.75 -0.041 -0.276 0.195 YES YES

316 86 323 117 203 5.56 -0.079 -0.314 0.156 YES YES

74 234 272 34 144 5.69 0.058 -0.177 0.293 YES YES

Reference method

Alternative method



fruit and vegfresh produce

Final ALSD repeatability of reference method <= 0.125

YES

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Bias

Reference Median

fresh produce

Bias

β-ETI

AL = +/- 0.5

Figure 4 Meat and poultry

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E.Coli:


β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

206 345 33 210 48 2.43 -0.016 -0.286 0.254 YES YES

90 81 293 260 286 2.74 0.052 -0.218 0.322 YES YES

185 199 26 208 178 4.46 0.000 -0.270 0.270 YES YES

280 220 14 311 50 4.57 0.095 -0.175 0.365 YES YES

335 191 99 19 193 6.73 0.060 -0.210 0.330 YES YES

336 237 84 278 221 6.70 0.114 -0.156 0.384 YES YES

Reference method

Alternative method



NO

(Food) Type fresh meat

Final AL

raw poultry and meat(Food) Category

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Bias

Reference Median

fresh meat

Bias

β-ETI

AL = +/- 0.5

Coliforms:

Sample Name Reference Central value

Bias Lower β-ETI Upper β-ETI

β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

168 135 283 214 47

1.88 -0.030 -0.411 0.351 YES YES

204 253 207 76 156

3.67 -0.188 -0.569 0.193 NO YES

284 225 85 257 96 4.37 -0.038 -0.419 0.343 YES YES

348 171 65 281 151

4.85 -0.340 -0.721 0.041 NO YES

167 36 324 216 133

5.04 0.163 -0.218 0.544 NO YES

165 69 310 118 349 6.51 -0.107 -0.488 0.274 YES YES

Reference method

Alternative method



NO

(Food) Type fresh meat

Final AL

raw poultry and meat(Food) Category

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Bias

Reference Median

fresh meat

Bias

β-ETI

AL = +/- 4SDr

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Figure 5 Ready to eat foods E.Coli:


β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

51 247 218 87 287 2.44 -0.062 -0.279 0.154 YES YES

163 241 152 227 125 2.48 0.270 0.054 0.487 YES YES

289 68 23 309 226 4.15 0.109 -0.107 0.326 YES YES

24 325 166 70 71 4.49 0.295 0.078 0.511 NO YES

195 261 42 61 320 5.79 0.203 -0.013 0.420 YES YES

62 145 266 317 271 6.00 0.322 0.106 0.539 NO YES

Reference method

Alternative method



NO


RTE foodscooked fish

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Bias

Reference Median

cooked fish

Bias

β-ETI

AL = +/- 4SDr

Coliforms:


β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

51 247 218 87 287 2.38 -0.050 -0.275 0.175 YES YES

111 155 255 186 202 2.66 0.267 0.041 0.492 YES YES

289 68 23 309 226 4.26 0.143 -0.083 0.368 YES YES

256 192 295 16 298 4.66 0.267 0.041 0.492 YES YES

195 261 42 61 320 6.00 -0.066 -0.291 0.159 YES YES

319 189 78 82 182 6.41 0.384 0.159 0.610 NO YES

Reference method

Alternative method


(Food) Category RTE foods

NO


(Food) Type cooked fish

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Bias

Reference Median

cooked fish

Bias

β-ETI

AL = +/- 4SDr

Figure 6 Multi component foods:

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E.Coli:

é


β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

37 20 28 148 126 2.23 -0.092 -0.271 0.087 YES YES122 169 282 60

159 2.32 0.211 0.032 0.390 YES YES

308 235 239 179 29 4.11 0.266 0.087 0.445 YES YES

113 318 112 279 27 4.20 0.075 -0.104 0.254 YES YES

251 128 329 30 146 6.15 0.234 0.055 0.413 YES YES

265 93 101 302 114 6.20 -0.028 -0.207 0.151 YES YES

Reference method

Alternative method


Mulit componentfood with raw ingredients



NO

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Bias

Reference Median

food with raw ingredients

Bias

β-ETI

AL = +/- 0.5

Coliforms:

é


β-ETI compared to

AL=±0.5 Acceptable

β-ETI compared to

final AL Acceptable

329 30 146 275 2422 1.60 0.243 0.003 0.483 YES YES

180 304 306 258 55 1.85 0.155 -0.085 0.395 YES YES

242 184 211 334 331 3.71 0.033 -0.207 0.272 YES YES

18 175 1 143 138 3.86 0.138 -0.101 0.378 YES YES131 59 161 103

209 5.61 0.086 -0.153 0.326 YES YES

245 115 224 249 315 5.95 -0.086 -0.326 0.154 YES YES

Reference method

Alternative method



NO

Mulit componentfood with raw ingredients


-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Bias

Reference Median

food with raw ingredients

Bias

β-ETI

AL = +/- 0.5

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The observed profiles are within the 0.5 log AL or the recalculated AL limit calculated according to ISO16140-2:2015 section 6.1.3.3. For both E.coli and coliforms, the accuracy profiles fulfil the performance criteria after the permitted recalculation and the alternative method is accepted as being equivalent to the reference methods. SELECTIVITY (INCLUSIVITY/EXCLUSIVITY) Inclusivity is the ability of an alternative method to detect the target analyte from a wide range of strains. For E.coli: In the original study: 31 strains were studied. All 31 strains grew and produced typical colonies on the Compact Dry EC medium. By comparison, 5 strains failed to grow in the TBX medium (ISO16649-2:2001) and one strain yielded atypical colonies. In the renewal study of 2018: Of the 20 inclusivity strains tested all strains were detected using both the alternative and reference method. For coliforms: In the original study: All 33 coliform strains produced typical colonies in VRBA (ISO 4832) and Compact Dry EC medium. In the renewal study from 2018: Of the 20 inclusivity strains tested 18 strains were detected using the alternative and the reference methods. Those not detected by either method were Shimwellia blattae NCTC 12127 and Klebsiella rhinoscleromatis CRA 4272. Exclusivity is the lack of interference from a relevant range of non-target strains of the alternative method. For E.coli: In the original study: The results from the 21 strains of non-target organisms showed that the majority (19 cultures) failed to grow or produced atypical colonies by both methods. Two strains of Shigella did yield typical colonies by both methods, which is not surprising because strains of Shigella have β-glucuronidase activity which would give rise to typical conies with chromogenic media developed to show this activity. In the current study (2018): Of the 10 exclusivity strains tested, none were detected by either the alternate or reference methods. For coliforms: In the original study: The results from the 20 strains of non-target organisms used to determine the exclusivity of the EC method showed that 9 strains did not grow on either the EC medium or on VRBA. In addition, one strain of Yersinia enterocolitica did not grow on the Compact Dry EC medium but did grow in VRBA. For Compact Dry EC, there were 7 strains giving atypical growth and 3 giving typical growth. For VRBA there were 5 strains giving atypical growth and 6 giving typical growth. In the recent study (2018): Of the 10 exclusivity strains tested, three were detected by the alternate method and by the reference method these were A.hydrophila CRA 4111, A.sobria CRA 8390 and S. fonticola CRA 4613.

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CONCLUSION OF THE COMPARISON STUDIES For E.coli: The results of the method comparison study showed that the Compact Dry EC provide equivalent results to the reference method ISO 16649-2:2001. For coliforms: The results of the method comparison study showed that the Compact Dry EC provide equivalent results to the reference method ISO 4832:2006. INTERLABORATORY STUDY OF E.COLI: The interlaboratory study was conducted in November 2007. Number of laboratories: 9 [13 labs participated. 2 were excluded as the analysis were not performed on the agreed date and further 2 labs failed to test their samples for E.coli by the reference method.] Samples: Pasteurised milk artificially contaminated with defined numbers of E.coli. The laboratories performed the analyses according to ISO 16649-2:2001 and Compact Dry EC method. Table 6 Results (log cfu/g) – interlaboratory study of E.coli

Referance method Alternative method Upper Lower Upper Lower Level Median SR Alt method SR Bias Level Level AL AL

1 2.40 0.17 2.45 0.18 0.05 0.30 -0.04 0.50 -0.50 2 3.50 0.17 3.46 0.17 -0.04 0.20 -0.17 0.50 -0.50 3 4.38 0.34 4.50 0.20 0.12 0.40 0.01 0.50 -0.50

The results show that the bias is small and that the precision is satisfactory. Figure 7 Accuracy Profile of the interlaboratory study for E.coli

According to the comparison study and the interlaboratory study no substantial differences were found between the Compact Dry EC method and the reference method (ISO 16649-2:2001) for the enumeration of Escherichia coli.

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INTERLABORATORY STUDY OF COLIFORMS The interlaboratory study was conducted in November 2007. Number of laboratories: 11 Samples: Pasteurised milk artificially contaminated with defined numbers of E.coli. The laboratories performed the analyses according to ISO 4832:2006 and Compact Dry EC method. Table 7 Results (log cfu/g) – interlaboratory study of coliforms

Referance method Alternative method Upper Lower Upper Lower Level Median SR Alt method SR Bias Level Level AL AL

1 2.53 0.16 2.55 0.13 0.02 0.20 -0.09 0.50 -0.50 2 3.59 0.11 3.57 0.19 -0.02 0.25 -0.10 0.50 -0.50 3 4.48 0.075 4.59 0.15 0.11 0.32 0.00 0.50 -0.50

The results show that the bias is small and that the precision is satisfactory. Figure 8 Accuracy Profile of the interlaboratory study for coliforms

According to the comparison study and the interlaboratory study no substantial differences were found between the Compact Dry EC method and the reference method (ISO 4832:2006) for the enumeration of coliforms.

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